I have designed a few pairs of sgRNA oligos using both CHOPCHOP and CRISPOR. I checked for their alignments within themselves. However, when I did the Primer-BLAST using both oligos (excluding the flanking sticky sequences). Only 1 of the 4 oligo pairs showed the gene of interest without any mismatch. All other pairs showed mismatch with non-specific genes, ironically these were ranked in the top in the sgRNA tool result. What may be the reason for mismatches and non-specific alignment? Please suggest.
Hi. In order to avoid off targets of the CRISPR system it is important to do the sgRNA BLAST.
I recommend choosing guides without any missmatch with the target gene, since the presence of any of them would decrease the editing efficiency. You also need to analyse where the off-target’s missmatches are: if they are on 3’ maybe it won’t be a problem, but if they are on 5’ you should take care as the 3’ will hybridize correctly with a non target gene.
It depends on if your specific organisms genome is in the NCBI database. If yes, that could work. It's tricky because you really need an exact match. What was the input for the sgRNA design programs you used? Just your favorite gene(s)?
So, the design programs will have limitations. If your genome has lots of similarity to your favorite gene it may be tricky to find sgRNAs without similarity to other genes/regions. What organism do you work in?
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